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Mechanisms of hyperhomocysteinemia induced skeletal muscle myopathy after ischemia in the CBS-/+ mouse model.

Veeranki S, Tyagi SC - Int J Mol Sci (2015)

Bottom Line: Although skeletal muscles express the key enzyme (MTHFR) that participates in re-methylation of Hcy into methionine, lack of trans-sulfuration enzymes (CBS and CSE) make skeletal muscles more susceptible to the HHcy-induced myopathy.Our study indicates that elevated Hcy levels in the CBS-/+ mouse skeletal muscles caused diminished anti-oxidant capacity and contributed to enhanced total protein as well as PGC-1α specific nitrotyrosylation after ischemia.Altogether these results suggest that HHcy exerts its myopathic effects via reduction of the PGC-1/PPARγ axis after ischemia.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology & Biophysics, University of Louisville, Louisville, KY 40202, USA. s0veer02@louisville.edu.

ABSTRACT
Although hyperhomocysteinemia (HHcy) elicits lower than normal body weights and skeletal muscle weakness, the mechanisms remain unclear. Despite the fact that HHcy-mediated enhancement in ROS and consequent damage to regulators of different cellular processes is relatively well established in other organs, the nature of such events is unknown in skeletal muscles. Previously, we reported that HHcy attenuation of PGC-1α and HIF-1α levels enhanced the likelihood of muscle atrophy and declined function after ischemia. In the current study, we examined muscle levels of homocysteine (Hcy) metabolizing enzymes, anti-oxidant capacity and focused on protein modifications that might compromise PGC-1α function during ischemic angiogenesis. Although skeletal muscles express the key enzyme (MTHFR) that participates in re-methylation of Hcy into methionine, lack of trans-sulfuration enzymes (CBS and CSE) make skeletal muscles more susceptible to the HHcy-induced myopathy. Our study indicates that elevated Hcy levels in the CBS-/+ mouse skeletal muscles caused diminished anti-oxidant capacity and contributed to enhanced total protein as well as PGC-1α specific nitrotyrosylation after ischemia. Furthermore, in the presence of NO donor SNP, either homocysteine (Hcy) or its cyclized version, Hcy thiolactone, not only increased PGC-1α specific protein nitrotyrosylation but also reduced its association with PPARγ in C2C12 cells. Altogether these results suggest that HHcy exerts its myopathic effects via reduction of the PGC-1/PPARγ axis after ischemia.

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Related in: MedlinePlus

Western blot images showing the levels of PPARγ, nitrotyrosine and PGC-1α in the eluates of PGC-1specific immunoprecipitation from different treatment groups of C2C12 cell lysates. GAPDH indicates input levels for the immune-precipitation experiments across the groups. The levels of PPARγ were also probed in the total lysates and are not significantly different (data not shown).
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ijms-16-01252-f006: Western blot images showing the levels of PPARγ, nitrotyrosine and PGC-1α in the eluates of PGC-1specific immunoprecipitation from different treatment groups of C2C12 cell lysates. GAPDH indicates input levels for the immune-precipitation experiments across the groups. The levels of PPARγ were also probed in the total lysates and are not significantly different (data not shown).

Mentions: To further understand the consequences of PGC-1α protein nitrotyrosylation and the conditions that favor protein nitrotyrosylation, we used the in vitro C2C12 myoblast model cell line. Previous study showed that NO donor SNP is toxic to C2C12 cells [27]. In light of this finding, we used a dose of SNP (30 μM) that is non-toxic to the cells in a 24 h period. All of our treatments did not produce any significant change in the cell morphology of differentiated C2C12 cells after the 24 h treatment period (data not shown). Differentiated C2C12 cells were treated with homocysteine or its cyclized metabolite homocysteine thiolactone (HcyTL) in the presence of nitric oxide donor SNP for 24 h. Cell lysates were assessed for total protein nitrotyrosine levels, as well as specific protein nitrotyrosine levels on PGC-1α. As show in Figure 5A,B, there was relatively increased nitrotyrosylation after Hcy or HcyTL treatment in the presence of NO donor SNP. Furthermore, there were increased nitrotyrosine levels on immunoprecipitated PGC-1α upon Hcy or HcyTL treatment in the presence of NO donor SNP (Figure 6). In addition, apparently there was an inverse relation between the associated PPARγ and the level of nitrotyrosylation present on the PGC-1α (Figure 6) after the PGC-1α specific pull-down. Given that the treatments of C2C12 cells did not significantly alter levels of PPARγ (Figure 6), reduced PPARγ-mediated downstream gene expression (as measured earlier for VEGF, [19]) coupled with its reduced association with PGC-1γ, together indicates that HHcy exerts its myopathic effects via reduction of the PGC-1α/PPARγ axis after ischemia through enhanced protein nitrotyrosylation.


Mechanisms of hyperhomocysteinemia induced skeletal muscle myopathy after ischemia in the CBS-/+ mouse model.

Veeranki S, Tyagi SC - Int J Mol Sci (2015)

Western blot images showing the levels of PPARγ, nitrotyrosine and PGC-1α in the eluates of PGC-1specific immunoprecipitation from different treatment groups of C2C12 cell lysates. GAPDH indicates input levels for the immune-precipitation experiments across the groups. The levels of PPARγ were also probed in the total lysates and are not significantly different (data not shown).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307302&req=5

ijms-16-01252-f006: Western blot images showing the levels of PPARγ, nitrotyrosine and PGC-1α in the eluates of PGC-1specific immunoprecipitation from different treatment groups of C2C12 cell lysates. GAPDH indicates input levels for the immune-precipitation experiments across the groups. The levels of PPARγ were also probed in the total lysates and are not significantly different (data not shown).
Mentions: To further understand the consequences of PGC-1α protein nitrotyrosylation and the conditions that favor protein nitrotyrosylation, we used the in vitro C2C12 myoblast model cell line. Previous study showed that NO donor SNP is toxic to C2C12 cells [27]. In light of this finding, we used a dose of SNP (30 μM) that is non-toxic to the cells in a 24 h period. All of our treatments did not produce any significant change in the cell morphology of differentiated C2C12 cells after the 24 h treatment period (data not shown). Differentiated C2C12 cells were treated with homocysteine or its cyclized metabolite homocysteine thiolactone (HcyTL) in the presence of nitric oxide donor SNP for 24 h. Cell lysates were assessed for total protein nitrotyrosine levels, as well as specific protein nitrotyrosine levels on PGC-1α. As show in Figure 5A,B, there was relatively increased nitrotyrosylation after Hcy or HcyTL treatment in the presence of NO donor SNP. Furthermore, there were increased nitrotyrosine levels on immunoprecipitated PGC-1α upon Hcy or HcyTL treatment in the presence of NO donor SNP (Figure 6). In addition, apparently there was an inverse relation between the associated PPARγ and the level of nitrotyrosylation present on the PGC-1α (Figure 6) after the PGC-1α specific pull-down. Given that the treatments of C2C12 cells did not significantly alter levels of PPARγ (Figure 6), reduced PPARγ-mediated downstream gene expression (as measured earlier for VEGF, [19]) coupled with its reduced association with PGC-1γ, together indicates that HHcy exerts its myopathic effects via reduction of the PGC-1α/PPARγ axis after ischemia through enhanced protein nitrotyrosylation.

Bottom Line: Although skeletal muscles express the key enzyme (MTHFR) that participates in re-methylation of Hcy into methionine, lack of trans-sulfuration enzymes (CBS and CSE) make skeletal muscles more susceptible to the HHcy-induced myopathy.Our study indicates that elevated Hcy levels in the CBS-/+ mouse skeletal muscles caused diminished anti-oxidant capacity and contributed to enhanced total protein as well as PGC-1α specific nitrotyrosylation after ischemia.Altogether these results suggest that HHcy exerts its myopathic effects via reduction of the PGC-1/PPARγ axis after ischemia.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology & Biophysics, University of Louisville, Louisville, KY 40202, USA. s0veer02@louisville.edu.

ABSTRACT
Although hyperhomocysteinemia (HHcy) elicits lower than normal body weights and skeletal muscle weakness, the mechanisms remain unclear. Despite the fact that HHcy-mediated enhancement in ROS and consequent damage to regulators of different cellular processes is relatively well established in other organs, the nature of such events is unknown in skeletal muscles. Previously, we reported that HHcy attenuation of PGC-1α and HIF-1α levels enhanced the likelihood of muscle atrophy and declined function after ischemia. In the current study, we examined muscle levels of homocysteine (Hcy) metabolizing enzymes, anti-oxidant capacity and focused on protein modifications that might compromise PGC-1α function during ischemic angiogenesis. Although skeletal muscles express the key enzyme (MTHFR) that participates in re-methylation of Hcy into methionine, lack of trans-sulfuration enzymes (CBS and CSE) make skeletal muscles more susceptible to the HHcy-induced myopathy. Our study indicates that elevated Hcy levels in the CBS-/+ mouse skeletal muscles caused diminished anti-oxidant capacity and contributed to enhanced total protein as well as PGC-1α specific nitrotyrosylation after ischemia. Furthermore, in the presence of NO donor SNP, either homocysteine (Hcy) or its cyclized version, Hcy thiolactone, not only increased PGC-1α specific protein nitrotyrosylation but also reduced its association with PPARγ in C2C12 cells. Altogether these results suggest that HHcy exerts its myopathic effects via reduction of the PGC-1/PPARγ axis after ischemia.

Show MeSH
Related in: MedlinePlus